Reciprocal interaction between lung mesenchymal and epithelial cells is essential for normal lung development and homeostasis. Therefore, alterations in lung mesenchymal cell biology will have a significant impact on both fetal lung formation and postnatal lung injury repair, resulting in the pathogenesis of many lung diseases. Although several sophisticated mouse genetic approaches have been obtained to study lung epithelial cell biology, the lack of lung mesenchymal cell-specific mouse genetic approaches to date has become a critical barrier to the field of lung research. We hypothesize that Tbx4 lung enhancer driven rtTA (reverse tetracycline- dependent transactivator) transgenic mouse line (Tbx4-rtTA) can be used to make a novel lung mesenchymal- specific Tet-On genetic system to study lung mesenchymal cell signaling in lung development and injury repair. Thus, combination of Tbx4-rtTA and TetO promoter driven dominant-negative TGF- receptor II (dnT2RII) transgenic lines will provide a unique tool to study TGF- signaling specifically in lung mesenchyme and its impact on lung development in vivo. Specific Aim 1. To develop and characterize a lung-mesenchymal cell-specific Tet-On genetic system. A 5.5 kb lung enhancer DNA element of Tbx4 gene will be used to make the Tbx4-rtTA transgenic mice. The lung mesenchymal localization of rtTA transgenic expression at different ages will then be verified. Furthermore, a lung mesenchymal cell-specific Tet-On genetic system will be generated and tested in mice by crossing Tbx4-rtTA and TetO-Cre mice with a floxed-mT-mG fluorescence protein reporter system. Specific Aim 2. To dissect the regulatory functions of TGF- signaling in developing lung mesenchymal cells in vivo using the established lung mesenchymal Tet-On genetic approach. We will apply the newly developed lung mesenchymal Tet-On system to characterize TGF- signaling in lung mesenchymal cells, as TGF- signaling plays extremely important roles in regulating lung development, injury repair, and lung diseases. By generating a Tbx4-rtTA/TetO-dnT2RII mouse line, lung mesenchymal-specific TGF- signaling can be reversibly inhibited at various levels and stages in vivo through dnT2RII induction. The roles of mesenchymal TGF- signaling in regulating lung development will then be dissected. Development of these novel genetic approaches has the potential to substantially extend current knowledge of lung mesenchymal cell biology and its relationship to lung diseases. Furthermore, by applying this system to lung mesenchymal TGF- signaling research, we will be able to dissect the genetic dosages and time windows of TGF- signaling in regulating lung development and maturation. Successful establishment of these unique genetic systems will provide powerful tools to define the role of mesenchymal signaling in lung development and homeostasis, and in the pathophysiology of lung diseases. Additionally, new therapeutic targets may be identified.